Resistant-starch formation in high-amylose maize starch
A new public high-amylose maize line, GEMS-0067, has been developed by USDA-ARS Germplasm Enhancement of Maize (GEM) Project. GEMS-0067 maize line is a homozygous mutant of amylose-extender (ae) gene and high-amylose modifier (HAM) gene(s). GEMS-0067 starches consist of 83.1-85.6% apparent amylose and 39.4%-43.2% resistant starch (RS), which are larger than the ae single-mutant starches (61.7-67.7% and 11.5%-19.1%, respectively) and normal maize starch (~30% and <1%, respectively). Maize ae-mutant starches consisted of spherical and elongated granules (up to 32%), which differed from normal maize starch, consisting of spherical and angular granules. The objectives of this study were to understand the mechanism of the RS formation in the maize ae-mutant starches, the crystalline structures of the maize ae-mutant starches, the formation of RS and elongated starch granules during the kernel development, and how dosage of the HAM gene(s) affected the structures and properties of endosperm starch of maize ae-mutant. We analyzed the structures and properties of the RS residues that remained after enzymatic hydrolysis of the maize ae-mutant starches at 95-100C (AOAC method 991.43). The RS residues consisted of mainly partially hydrolyzed amylose and intermediate component (IC) and were more concentrated in the elongated starch granules and the outer layer of the spherical granules. The RS residues displayed the B-type polymorph and had gelatinization temperatures above 100C. These results suggested that long-chain double-helical crystallites of amylose/IC were present in the native maize ae-mutant starches. The amylose/IC crystallites maintained the semi-crystalline structures at 95-100C and were resistant to enzymatic hydrolysis at 95-100C.
To understand the crystalline structures of the maize ae-mutant starches, we prepared Naegeli dextrins of the starches using sulfuric acid (15.3%, v/v) hydrolysis of the granular starch at 38C for up to 102 days. The yields of the Naegeli dextrins ranged from 18.3 to 39.5%. The Naegeli dextrins displayed similar onset (45.1-51.4oC), peak (113.9-122.2oC), and conclusion (148.0-160.0oC) gelatinization temperatures and had large enthalpy-changes (21.8-31.3 J/g) and percentage crystallinity (77.0-79.2%). The Naegeli dextrins showed unimodal molecular-size distributions with peak molecular-size at degree of polymerization (DP) 16. The molecular-size distributions of the Naegeli dextrins did not significantly change after debranching with isoamylase, indicating that the Naegeli dextrins are composed predominantly of linear molecules. The isoamylase-debranched Naegeli dextrins had average chain-lengths of DP 23.8-27.5 and large proportions of long chains (DP 25, 36.7-52.7%), resulting from hydrolysis of amylose double helices.
The RS contents and physicochemical properties of the ae-mutant maize (GEMS-0067) starches harvested at different kernel-developmental stages were analyzed. The RS content increased with kernel maturation and the increase in the amylose/IC content. The formation of long-chain double-helical crystallites of amylose/IC and the lipid content increased with kernel maturation and the increase in the amylose/IC content. These results suggested that the increase in the long-chain double-helical crystallites of amylose/IC and the increase in lipid content resulted in the increase in the RS content of the GEMS-0067 starch during the kernel development
The light and confocal laser-scanning micrographs of the physiologically mature GEMS-0067 starch granules showed that the arrangement of starch molecules varied between granules and within a granule. The transmission electron microscopic images of GEMS-0067 endosperm tissues harvested at an early stage (20 days after pollination) of the kernel development showed that the elongated starch granules formed by fusion of small granules through amylose interaction in the amyloplast at the early stage of granule development.
To understand how dosage of the high-amylose modifier (HAM) gene affected the structures and properties of endosperm starch of maize ae-mutant, nine maize samples with HAM gene-dosage of 100%, 83.3%, 66.7%, 66.7%, 50%, 33.3%, 33.3%, 16.7%, and 0% were prepared in a generation-means analysis (GMA) study. An increase of HAM gene-dosage in maize ae-mutant resulted in higher amylose/IC content of the starch and significantly affected the molecular organization in the granule, granule morphology, starch crystallinity, and starch thermal properties. RS content of the starch was significantly correlated with HAM gene-dosage (r = 0.81, P 0.01). The increase in HAM gene dosage in the endosperm of ae-mutant maize had little effect on the branch chain-length of the amylopectin and large molecular-weight IC but increased branch chain-length of the small molecular-weight IC. The HAM gene dosage also had little effect on the structure of amylose of the maize ae-mutant starch.